Biology subjects

Tolosa, E.

Publications and source records attributed to Tolosa, E..

9 recordsLinked to original sources

bioRxiv2025

Phospho-signalling deregulation of the mTOR pathway in fibroblasts from LRRK2 Parkinson s patients

Activating mutations at the leucine-rich repeat kinase 2 (LRRK2) gene are the most frequent genetic cause of Parkinsons Disease (PD). Using in-depth mass-spectrometry, here we analysed the impact of LRRK2 mutations on the proteome and phospho-proteome of skin fibroblasts from a large LRRK2 clinical cohort from Spain. The cohort encompassed G2019S LRRK2-associated PD (L2PD) (n=15), G2019S LRRK2 non-manifesting carriers (L2NMCs) (n=13), idiopathic PD (iPD) (n=12), and control subjects (n=14) (total n=54). The proteome analysis revealed 89 differential proteins in G2019S L2PD (58 down/ 31 up) and 168 in G2019S L2NMCs (127 down / 41 up) compared to controls. Most of these proteome changes involved deficits in proteins related to mitochondrial energy and ribosomal protein synthesis. At the phospho-proteome level, we identified 394 differential phospho-sites in G2019S L2PD (188 hyper / 206 hypo), 367 in G2019S L2NMCs (215 hyper / 152 hypo), and 428 in iPD (214 hyper / 214 hypo) compared with controls. These phospho-proteomic changes included hyper-phosphorylated proteins associated with the mTOR and Rho GTPase pathways. Notably, the top hyper-phosphorylated hit in G2019S carriers was pThr37/46 4E-BP1, a key effector of the mTOR pathway controlling protein synthesis, and this protein was also deregulated in iPD. In summary, our study uncovers peripheral deregulation of mTOR phospho-signalling associated with LRRK2 mutations, which is related to deficits in mitochondrial and ribosomal proteins, and affects converging pathways altered in iPD.

neuroscience↗

bioRxiv2025

Defective proviruses cause T cell reprogramming through promoter exaptation in HIV-1 infection

People living with HIV (PLWH) on antiretroviral therapy (ART) accumulate primarily defective proviral sequences in genomes of often clonally expanded CD4+ HIV-1 target cells. The majority of viral-derived DNA is transcriptionally active and preferentially found at distinct genomic loci suggesting a selective process driven by integration site-specific crosstalk between viral and host sequences. Focusing on one of the most prominent selected integration loci, the BTB Domain and CNC Homolog 2 (BACH2) gene, we here show mechanistic insights how CD4+ T cells are functionally reprogrammed via exaptation of provirus-derived regulatory sequences during long-term ART. Using a cellular model of BACH2-integrated proviruses, we find that proviral transcription drives aberrant BACH2 protein levels that escape autoregulatory feedback and impose BACH2-dependent transcriptomic changes. By mimicking these changes in primary CD4+ T lymphocytes, we observe that BACH2 drives reprogramming of cells toward a proliferative, precursor memory-like type. These reprogrammed CD4+ T cells possess traits of immune evasion and cellular survival that are signatures of persistent HIV reservoir cells in PLWH. Inhibition of provirus transcriptional activity can mitigate exaptation, suggesting a strategy to offset HIV-driven differentiation and expansion of CD4+ T cells. Finally, our data suggest that provirus exaptation at a second prominently selected proviral integration gene, the Signal Transducer And Activator of Transcription 5B (STAT5B) gene, drives a contrary, effector-like T cell fate, suggesting a multifaceted impact of exaptation on immune homeostasis. Overall, our data suggest that transcriptionally active proviruses, even if structurally defective, modulate target cells through insertional activation of integration genes, a process which we postulate to contribute to the complex immune modulation and dysregulation experienced by ART-suppressed PLWH.

microbiology↗

bioRxiv2025

Spatial Analysis of T Cell Clonality in Autoimmune Kidney Disease Using TRV Probes

Hypothesizing that the localization of T cell clones correlates with immune function, our goal was to develop an unbiased method to study the spatial distribution of T cells in tissues. We created an in situ hybridization panel with 248 probes that identify immune and tissue cell types, and 132 probes for all variable TRAV, TRBV, TRGV, and TRDV gene segments. Applying this approach to analyze renal biopsies from patients with autoimmune kidney disease, combinations of TRV segments provided spatial information about T cell clonality. Confined clusters of clonally related {beta} T cells were found in proximity to increased numbers of antigen-presenting cells, B cells, and other T cells, reflecting local immune cell interactions. {gamma}{delta} T cells were more frequently located outside or at their periphery of T cell infiltration areas. In conclusion, integrating spatial information with TCR clonotype analysis provided new insights into the organization of immune responses at the tissue level.

immunology↗

bioRxiv2024

Fluorescence based live cell imaging identifies exon 14 skipped hepatocyte growth factor receptor (MET) degraders

Despite ongoing efforts to employ structure-based methods to discover targeted protein degraders (TPD), the prevailing strategy continues to be the synthesis of a focused set of heterobifunctional compounds and screen them for target protein degradation. Here we used a fluorescence based live cell imaging screen to identify degraders that target exon 14 skipped hepatocyte growth factor receptor (MET). MET is a known oncogenic driver. MET exon 14 skipping mutations (METex14{Delta}) are found in lung cancers and result in the loss of a degron that is required for E3-ligase recognition and subsequent ubiquitination, prolonging the half-life and oncogenicity of MET. Since proteolysis targeting chimeras (PROTACs) are heterobifunctional molecules that promote target degradation by the proteosome, we sought to restore degradation of MET lost with METex14{Delta} using a MET-targeting PROTAC. We generated a library of sixty PROTACs of which 37 used the MET inhibitor capmatinib as the protein of interest targeting ligand. We screened this PROTAC library for targeted degradation of METex14{Delta}-GFP using live cell imaging. We benchmarked out MET-targeting PROTACs to that of a previously reported MET-targeting PROTAC, SJF8240. Curve fitting live cell imaging data affords determination of time required to degrade 50% of the target protein (DT50), which was used in determining structure activity relationships. A promising candidate, 48-284, identified from the screen, exhibited classic PROTAC characteristics, was > 15-fold more potent than SJF8240, had fewer off targets compared to SJF8240, and degraded MET in multiple cell lines.

cancer biology↗

bioRxiv2024

LRRK2-mutant microglia trigger dopaminergic neurodegeneration when activated by neuromelanin

Parkinsons disease (PD) is a progressive, incurable neurodegenerative disorder characterized by the loss of neuromelanin (NM)-containing dopamine neurons (DAn) in the substantia nigra of the midbrain. Non-neuronal cells are increasingly recognized as contributors to PD. We generated human microglia-like cells (hMG) from induced pluripotent stem cells (iPSC) derived from patients with LRRK2 PD-causing mutations, gene-corrected isogenic controls, and healthy donors. While neither genotype induced neurodegeneration in healthy DAn, LRRK2 hMG become hyperreactive to LPS stimulation, exhibiting increased cytokine expression, reactive oxygen species, and phagocytosis. When exposed to NM-containing particles, but not -synuclein fibrils, LRRK2 hMG trigger DAn degeneration, in a process that is prevented by pre-treatment with the immunomodulatory drug ivermectin. Finally, post-mortem analysis of midbrain tissue of LRRK2-PD patients show increased microglia activation around NM-containing neurons, confirming our in vitro findings. Overall, our work highlights NM-activated microglias role in PD progression, and provides a model for testing therapeutic targets.

cell biology↗

bioRxiv2024

Limited protection against early-life cytomegalovirus infection results from deficiency of cytotoxic CD8T cells

Differential antiviral T cell immunity in early life impacts the clinical outcome of Cytomegalovirus (CMV) infection, but the underlying mechanisms are not well understood. Here, we found delayed enrichment of early-life murine CMV-specific CD8 T cells due to a general deficiency of {beta} T cells. Adoptive transfer of naive adult T cells into neonates did not protect due to a blockade of CD8 but not of CD4 effector T cell differentiation. Early-life deficiency of critical signal 3 cytokines during T cell priming resulted in the appearance of non-cytotoxic CD8 effector T cells whereas the effector phase of adult-primed T cells was not disrupted in neonates. Accordingly, we found an overall low number of antiviral human CD8 T cells in newborns with congenital CMV. Together, this study suggests defective CD8 T cell immunity as an important factor explaining the higher risk for CMV disease in the early-life phase.

immunology↗

bioRxiv2024

NSCLC patients with oligo-metastatic brain disease show an altered CD4 T-cells immune profile

BackgroundLung cancer is the leading cause of cancer-related deaths worldwide, with brain metastasis (BM) occurring in 40% of advanced non-small cell lung cancer (NSCLC) patients. In 15% of these patients, the brain is the only affected organ (oligo-metastasis), corresponding to improved prognosis compared to widespread disease. Thus far, it is unknown if the metastatic dissemination to the brain without systemic metastases is a consequence of the immune systems ability to control systemic tumor outgrowth. MethodsHere, we investigated the local and peripheral immune cell composition in NSCLC BM patients, and identified new immune patterns related to the occurrence of brain metastases either as oligo- or poly-metastatic disease. ResultsThe multi-parametric immune phenotyping of peripheral blood revealed a downregulation of KLRG1 in CD8+ T-cells and an increase in CD4+ TH17 cells and elevated IL-17 levels in the blood of all NSCLC BM patients compared to healthy individuals. In addition, BM patients CD4+ T cells showed less CD73 expression with reduced effector memory differentiation. Furthermore, we observed less intra-tumoral infiltration in tumor tissues and a distinctive CD4+ T-cell profile in oligo-synchronous BM, both in the tumor microenvironment and peripheral blood compared to poly-metastatic BM patients. Moreover, 5'-ectonucleotidase CD73 was significantly upregulated in CD4 and T-regulatory cells of oligo-synchronous BM. ConclusionsThese results indicate that oligo-synchronous BM exhibits a more pronounced alteration in the CD4 T-cell immune profile both locally at the tumor site and systemically. Key PointsO_LIBM patients exhibit a skewed systemic immune profile, characterized by downregulation of KLRG1 in CD8+ and induction of TH17/IL-17 axis and CD73 in CD4+ T-cells. C_LIO_LIOligo-synchronous BM displayed a distinct CD4+ T-cell profile in both TME and peripheral blood. C_LI Importance of the StudyThis study presents a novel insight into immune profiles of brain metastasis types in NSCLC patients. Examining tissues and PBMCs sheds light on the disease and uncovers unique immune responses within distinct brain metastasis patterns. This research offers valuable knowledge for improved understanding and identifying potential prognosis markers.

cancer biology↗

bioRxiv2023

Cleavage site-directed antibodies reveal the prion protein in humans is shed by ADAM10 at Y226 and associates with misfolded protein deposits in neurodegenerative diseases

Proteolytic cell surface release ( shedding) of the prion protein (PrP), a broadly expressed GPI-anchored glycoprotein, by the metalloprotease ADAM10 impacts on neurodegenerative and other diseases in animal and in vitro models. Recent studies employing the latter also suggest shed PrP (sPrP) to be a ligand in intercellular communication and critically involved in PrP-associated physiological tasks. Although expectedly an evolutionary conserved event, and while soluble forms of PrP are present in human tissues and body fluids, neither proteolytic PrP shedding and its cleavage site nor involvement of ADAM10 or the biological relevance of this process have been demonstrated for the human body thus far. In this study, cleavage site prediction and generation (plus detailed characterization) of sPrP-specific antibodies enabled us to identify PrP cleaved at tyrosin 226 as the physiological and strictly ADAM10-dependent shed form in humans. Using cell lines, neural stem cells and brain organoids, we show that shedding of human PrP can be stimulated by PrP-binding ligands without targeting the protease, which may open novel therapeutic perspectives. Site-specific antibodies directed against human sPrP also detect the shed form in brains of cattle, sheep and deer, hence in all most relevant species naturally affected by fatal and transmissible prion diseases. In human and animal prion diseases, but also in patients with Alzheimers disease, sPrP relocalizes from a physiological diffuse tissue pattern to intimately associate with extracellular aggregates of misfolded proteins characteristic for the respective pathological condition. Findings and research tools presented here will accelerate novel insight into the roles of PrP shedding (as a process) and sPrP (as a released factor) in neurodegeneration and beyond.

neuroscience↗

bioRxiv2022

Immunosuppressive tocilizumab prevents astrocyte induced neurotoxicity in hiPSC-LRRK2 Parkinson's disease by targeting receptor interleukin-6

Parkinsons disease (PD) is associated with premature death of dopamine-producing neurons in the brain. Previous studies have shown that astrocytes of PD patients may contribute to neuronal degeneration by mechanisms involving both direct cell-to-cell contact and transfer of soluble molecules. Since it has been proposed that PD patients exhibit an overall pro-inflammatory state, and since astrocytes are key mediators of the inflammation response in the brain, here we sought to address whether astrocyte-mediated inflammatory signaling could contribute to PD neuropathology. For this purpose, we generated astrocytes from induced pluripotent stem cells (iPSCs) representing PD patients and healthy controls. Transcriptomic analyses identified a unique inflammatory gene expression signature in PD astrocytes compared to controls. In particular, the pro-inflammatory cytokine IL-6 was found to be highly expressed and released by PD astrocytes, and to induce toxicity in dopamine neurons. Mechanistically, neuronal cell death was mediated by IL-6 signaling via IL-6 receptor (IL-6R) expressed in human PD neurons, leading to downstream activation of STAT3. Importantly, astrocyte-induced cell death in PD disease midbrain neurons could be prevented by blocking IL6R-mediated signaling using clinically available antibodies. Moreover, examination of postmortem tissue brain of early-stage PD patients uncovered increased numbers of dopamine neurons overexpressing IL-6R and of reactive astrocytes overexpressing IL-6, compared to healthy brains. Our findings highlight the potential role of astrocyte-mediated inflammatory signaling in neuronal loss in PD, and open the way for new therapies based on IL-6 immunomodulation for preventing PD pathogenesis.

neuroscience↗